Our long-term goals are to understand the evolution of catabolic pathways and we are using the metabolism of 2,4-dichlorophenoxyacetic acid (2,4- D) as a model system. We are studying the changes that occur in soil microorganisms when exposed to repeated applications of 2,4-D allowing efficient degradation. Though these microorganisms degrade 2,4-D, the huge volume of 2,4-D used in the United States overwhelms the microorganisms causing pollution of water and soil. Exposure to 2,4-D is associated with health problems including liver, kidney and thyroid damage. We are interested in the role of regulatory elements in the changes that allow the microorganisms to degrade 2,4-D. Are regulatory elements assembled with structural genes they regulate? First will look at the regulatory elements of the 2,4-D pathway in a diverse group of 2,4-D degrading organisms that have been characterized with respect to enzyme function and DNA sequences. We will determine the degree of conservation for both the function and the DNA sequences of the regulatory elements. To find clues to how specific regulation is achieved we will examine the conservation of regulatory function in pathways for the metabolism of compounds chemically similar to 2,4-D, including other chloroaromatic compounds and the non-chlorinated forms of these compounds. We will also experimentally manipulate the regulatory loci to determine regulatory elements in adapting to new xenobiotics. Understanding how microorganisms evolve to degrade 2,4-D, a man- made chemical, may aid in the development of bioremediation strategies for 2,4-D and other xenobiotics and may provide information that could be used to engineer organisms that degrade of more recalcitrant and toxic pollutants.